Video 2. Representative OT-1 thymocyte signaling event.
Preselection OT-1 thymocytes were loaded with the ratiometric calcium indicator dye Indo1LR and allowed to migrate into selecting thymic slices for 6 hr, then imaged using two-photon-microscopy. (Left) Max z-projection of calcium high in magenta and calcium low in green. (Center) Max z-projection of the ratio of fluorescent signal in the calcium-bound channel over calcium-unbound channel for Indo1LR displayed as a heatmap (red = calcium high, purple = calcium low). (Right) Calcium (Ca2+) ratio and speed (averaged over two intervals) of the signaling cell. Arrow indicates time points in which the cell displays elevated calcium (230 s). Frames were collected every 10 s for 14.3 min. Dimensions: 68.9 μm (width) by 49.8 μm (height) by 18 μm (depth). Scale bar is 10 μm.